Estrogen (E2) has multiple and complex regulatory effects on T cell function which may be relevant for the pathogenesis of postmenopausal osteoporosis. However, a cause-effect relationship between T cell alterations and postmenopausal bone loss remains to be established. We found that ovariectomy (ovx) failed to induce bone loss in T cell deficient nude mice. The capacity of ovx to induce bone loss was restored by T cell reconstitution with Wild type T cells but not by constitution with T cells from TNF-/- mice. Moreover, ovx increased T cell TNF production by enhancing T cell proliferation and decreasing T cell apoptosis. These events led to an increase in the number of TNF producing T cells in the bone marrow. These findings demonstrated that T cells and T cell produced TNF are essential mediators of the bone-wasting effects of E2 deficiency in vivo. However, the specific phenotype of the E2 regulated TNF producing T cell remains to be characterized. Equally enigmatic are the mechanisms by which E2 down regulates the number of TNF producing T cells in the bone marrow. Therefore, our first Specific Aim will be to identify the specific T cell population (CD4+ CD8+ and/or gamma-delta T cells) that produces increased amounts of TNF and cause bone loss in ovx mice. The relevance of the candidate T cell population will be demonstrate by determining if ovx induces bone loss in mice lacking either CD4+, CD8+ and/or gamma-delta lymphocytes. In Specific Aim 2 we will determine if E2 blocks T cell dependent bone loss via a direct targeting of T cells or via effects on bone marrow accessory cells. This will be accomplished by analyzing the effects of ovx and E2 replacement on bone density, T cell proliferation, T cell apoptosis and T cell TNF production in nude mice reconstituted with T cell harvested from E2 receptor alpha and beta deficient donors. Our experimental evidence suggests that E2 blocks T cell TNF production by repressing IL-7, a cytokine which regulates T cell proliferation and induces bone loss. Therefore, we will evaluate the contribution of IL-7 by determining if in vivo neutralization of IL- 7 prevents the increase in T cell TNF production and the bone loss induced by ovx. Our data also suggest that E2 represses T cell TNF production by blunting IFN-gamma induced macrophage antigen presenting cell (A PC) activity, through down regulation of CIITA gene expression. Therefore, we will conclude Aim 2 by using IFN-gamma neutralization and IFN-gR1 -/- mice to determine the effects of CIITA silencing on T cell TNF production and bone loss. In specific Aim 3 we will investigate the mechanism which E2 represses CIITA gene expression. This will be accomplished by analyzing the effects of E2 on the transcription and the translation of CIIT A and by determining the Cisregulating factors that confer E2 responsiveness to the CIITA promoter. The significance of this project is high, as it may shed light on the pathogenesis of postmenopausal osteoporosis and identify novel target of E2 in bone.